Heterotrophic microbial activity in lake sediments:effects of organic electron donors

Isabela C. Torres • Kanika S. Inglett •

K. R. Reddy

Received: 27 July 2009 / Accepted: 9 June 2010

� Springer Science+Business Media B.V. 2010

Abstract Allochthonous and autochthonous organic

matter deposited in benthic sediments are mineralized

by microbial communities, resulting in release of

nutrients to the water column. Lakes with different

trophic states may have sediments with different carbon

and nutrient concentration with consequently different

microbial communities. Microbial diversity of surface

sediments of three subtropical lakes of different trophic

state was investigated by measuring catabolic response

to a wide variety of carbon-substrates. Basal carbon

dioxide and methane production rates were highest in

Lake Apopka (hypereutrophic), followed by Lake

Annie (oligo-mesotrophic) and Lake Okeechobee

(eutrophic) sediments. The oligo-mesotrophic Lake

Annie showed the highest metabolic quotient (qCO2;

proportion of basal respiration per unit of microbial

biomass, 0.008 ± 0.001) indicating inefficient use of

energy. The low qCO2 found in Lake Apopka sediment

indicated higher efficiency in using energy. Lake

Okeechobee sediments had intermediary values of

qCO2 (M9 0.005 ± 0.001; M17 0.006 ± 0.0003; KR

0.004 ± 0.001) as compared with other lakes (lake

Apopka 0.004 ± 0.14). Lake Apopka’s sediment cat-

abolic diversity was higher than that observed in other

sediments. Addition of organic electron donors to

sediment samples from all lakes stimulated heterotro-

phic activity; however, the extent of the response varied

greatly and was related to microbial biomass. The

hypereutrophic Lake Apopka sediments had the highest

respiration per unit of microbial biomass with the

addition of electron donors indicating that these

sediments respired most of the C added. These results

showed that sediments with different biogeochemical

properties had microbial communities with distinct

catabolic responses to addition of the C sources.

Keywords Electron donor � Microbial activity �Nutrients � Organic carbon � Sediment

Introduction

Organic matter deposition is an important source of

carbon (C) in lake sediments. Organic compounds and

associated nutrients supplied to sediments are miner-

alized through heterotrophic decomposition (Capone

and Kiene 1988; Megonigal et al. 2004). The compo-

sition and activities of microbial communities are

regulated by the quality and availability of carbon. In

high depositional environments such as eutrophic or

deep thermally stratified lakes, organic content in

sediments is often high, such that oxygen (O2) is rapidly

consumed, and is depleted within several millimeters

below the sediment water interface (Jørgensen 1983).

In these systems, facultative and obligate anaerobic

I. C. Torres � K. S. Inglett � K. R. Reddy (&)Wetland Biogeochemistry Laboratory, Soil and Water

Science Department, University of Florida, Newell Hall

106, Gainesville, FL 32611-0510, USA

e-mail: krr@ufl.edu

123

Biogeochemistry

DOI 10.1007/s10533-010-9494-6

communities dominate. Complete oxidation of a broad

range of organic compounds in these systems can occur

through the sequential activity of different groups of

anaerobic bacteria (Capone and Kiene 1988).

In anaerobic environments, cellulolytic bacteria

hydrolyze organic polymers through extracellular

enzyme production and further break down monomers

to alcohols, fatty acids, and hydrogen (H2) through

fermentation. Alcohols and fatty acids are then further

degraded by syntrophic bacteria (secondary ferment-

ers) into acetate, H2, and carbon dioxide (CO2), which

can be used as substrate by methanogens (Zinder 1993;

Conrad 1999). The structure and function of anaerobic

microbial communities are therefore strongly affected

by competition for fermentation products such as H2,

CO2 and acetate. Microorganisms derive energy by

transferring electrons from an external source or donor

to an external electron sink or terminal electron

acceptor. Range of organic electron donors vary from

monomers that support fermentation to simple com-

pounds such as acetate and methane (CH4). Ferment-

ing, syntrophic, methanogenic bacteria and most

other anaerobic microorganisms (e.g., sulfate and iron

reducers) are sensitive to the concentrations of

substrates and their activities can be inhibited by

accumulation of their end products. Consequently they

are dependent on the end product consumption by

other organisms (Stams 1994; Megonigal et al. 2004).

Measuring catabolic response profile has been

widely applied in soil studies as a method to charac-

terize the microbial functional diversity (Degens and

Harris 1997; Lu et al. 2000; Degens et al. 2000).

Substrate induced respiration (SIR) is often dependent

on the size of the microbial biomass pool; however,

response of microbial communities is also related to

the catabolic diversity of soil microorganisms (Degens

1998; Wright and Reddy 2001, 2007). A greater

catabolic response to a substrate in one system as

compared with another indicates that the microbial

community is more functionally adept in utilizing that

substrate and may indicate previous exposure to the

associated C-sources (Degens and Harris 1997; De-

gens 1998; Baldock et al. 2004).

The varied metabolic response of microbial com-

munities in lake sediments may also be related to the

physico-chemical characteristics of lakes (the source

and chemical composition of particulate matter and

biogeochemical processes in the sediment and the

water column). Accumulation and retention of

particulate matter and nutrients in sediments depends

on lake morphometry, water renewal, nutrient loading,

edaphic characteristics of the drainage basin, among

other factors (Boström et al. 1988). Eutrophic and

hypereutrophic lakes typically receive high external

loads of nutrients and display high primary produc-

tivity and nutrient concentrations in the water column.

Consequently, sediments from eutrophic and hype-

reutrophic lakes are expected to have high concentra-

tions of organic matter and associated nutrients. Net

accumulation rates of organic matter and nutrients

increase with trophic state of lakes (Binford and

Brenner 1986; Deevey et al. 1986). In contrast, small

oligotrophic lakes are expected to exhibit a relatively

high proportion of allochthonous carbon input to

sediments (Gu et al. 1996).

Lake depth can also affect the quality of organic

material reaching the sediment. In deep lakes, sedi-

menting organic matter undergoes intense decompo-

sition in the water column due to the prolonged period

of settling. Consequently, low amounts of labile

organic carbon reach the sediment in deep lakes

(Suess 1980; Meyers 1997). In shallow lakes, the

supply of labile organic C and nutrients can be higher

in sediments than in deep lakes, while deep lakes often

can have more refractory organic matter (Suess 1980).

Sediments receiving organic C of varying lability

can support different microbial communities. These

communities can display distinct catabolic responses

as the mineralization rates of a microbial community

are dependent upon the metabolic capacity for a

given substrate (Torien and Cavari 1982). The objec-

tives of this study were: (1) to evaluate the short-term

potential catabolic response of microbial communities

in sediments of three subtropical lakes characterized

with different trophic states, and (2) to examine

linkages between these microbial potential catabolic

responses and the physico-chemical nature of the

lakes. The central hypothesis was that sediments with

higher C availability would have higher catabolic

diversity.

Methods

Study sites

Three subtropical Florida lakes (USA) were selected

for this study based on water quality variables and

Biogeochemistry

123

trophic status (Table 1; Fig. 1). Lake Annie (Fig. 1a),

a small (0.37 km2) oligo-mesotrophic lake, is located

in south-central Florida (Highlands County) at the

northern end of the Archbold Biological Station.

Lake Annie is characterized by pristine water quality

with little surface water input (most is ground water),

and low anthropogenic impact due to the absence of

development around the lake (Layne 1979). This lake

has no natural surface streams but two shallow man

made ditches allow surface water to flow into the lake

and contribute to water and nutrient inputs during

high rainfall periods (Battoe 1985). Benthic sedi-

ments vary from organic to sand in the littoral zone

(Layne 1979). Lake Okeechobee (Fig. 1b) is a large

(1,800 km2) shallow lake located in south Florida. It

is considered to be a eutrophic lake that has

experienced cultural eutrophication over the past

50 years (Engstrom et al. 2006). Benthic sediments

include mud (representing 44% of the total lake

surface area), sand and rock (28%), littoral, domi-

nated by macrophyte growth (19%), and peat (9%)

that refers to partially decomposed plant tissues

(Fisher et al. 2001). Lake Apopka (Fig. 1c) is also a

shallow lake with 125 km2 surface area, located in

central Florida. Once a clear-water macrophyte-

dominated lake, since 1947 Lake Apopka has tran-

sitioned to a turbid, algal-dominated lake (Clugston

1963) considered hypereutrophic. This shift may

have been caused by nutrient input from several

sources, including agricultural drainage from adja-

cent vegetable farms (Schelske et al. 2000). Benthic

sediments are characterized by unconsolidated mate-

rial mainly consisting of algal deposits (Reddy and

Graetz 1991).

Field sampling

Triplicate intact sediment cores were collected using

a piston corer (Fisher et al. 1992) or by SCUBA

divers. The topmost 10 cm of sediment was collected

from one central site in Lake Annie on June 25, 2005

and a western site in Lake Apopka on May 28, 2005

(Fig. 1a, c; Table 1). Cores were collected at three

sites in Lake Okeechobee on July 16, 2005:

M17 = peat, M9 = mud, and KR = sand (Fig. 1b;

Table 1). Although there was a difference in the days

Table 1 Characteristics of sampled sites in the three different lakes with sampling date, location, sediment type and water qualityparameters (measured at 1 m)

Parameters Lake

Anniea Okeechobeeb Apopkab

Central M9 M17 KR West

Sampling date June/2005 July/2005 May/2005

Sediment type Mud/Clay Mud Peat Sand Organic

Water column depth (m) 20 4.0 2.5 3.1 2.0

Secchi (m) 2.0 0.08 0.15 0.5 0.3

Latitude 27�1202700 26�58017.600 26�45024.400 27�58011.500 28�3800100

Longitude 81�2104400 80�45038.400 80�46036.800 81�00051.800 81�3903600

Temperature (�C) 30.2 29.5 28.8 30.8 26.6Electrical Conductivity (lS cm-1) 42 385 320 143 443

pH 5.1 7.8 7.6 6.0 7.6

Dissolved oxygen (mg l-1) 6.4 6.5 6.3 1.8 8.7

Dissolved organic carbon (mg l-1)* 13.8 14.5 17.9 19.8 31.1

Total phosphorus (lg l-1)* 33 256 263 146 70

Dissolved reactive phosphorus (lg l-1)* 7 90 113 62 11

Total nitrogen (lg l-1)* 1,807 3,439 3,362 2,957 11,149

Ammonium—NH4–N (lg l-1)* 182 103 60 84 120

* Mean concentration in the water columna Average depth: 0.5, 1, 2, 5, 10, 20 (m)b Average depth: 0.5, 1, 2 (m)

Biogeochemistry

123

the samples were collected, all lakes were sampled

within the same season (summer time) when temper-

atures were high. So, the seasonality effect that can

occur in subtropical lakes would not interfere in the

outcome of the experiments. Sediment samples

(0–10 cm) were placed in sterile plastic sampling

bags, sealed, and kept on ice. All measured sediment

variables are reported on a dry weight basis (dw).

AB

C

0 50 100 150 200 250 sretemoliK52

0 0.1 0.2 0.3 Kilometers

(a) Lake Annie Florida

0 2 4 6 sretemoliK1

WestWest

(b) Lake Okeechobee (c) Lake Apopka

Fig. 1 Map of the three subtropical lakes with sampled sites and their locations in Florida State: a Lake Annie (with water columndepth in meters, modified from Layne 1979), b Lake Okeechobee with different sediment types, and c Lake Apopka

Biogeochemistry

123

Water samples were collected from various depths

(Table 1) at each site using a Van Dorn bottle and

several chemical parameters were measured to char-

acterize the lakes in relation to their trophic status.

Water transparency was determined using a Secchi

disk. Water temperature (�C), electrical conductivity(EC), pH, and dissolved oxygen (DO) were measured

with a YSI 556 Multi-Probe Sensor (YSI Environ-

mental, Yellow Springs, OH, USA) at different depths

(Table 1). Water column nutrient concentrations were

measured using U.S. EPA methods (EPA 1993). Total

Kjeldahl nitrogen (TKN) was measured by digestion

with concentrated sulfuric acid (H2SO4) and Kjeldahl

salt catalyst, and determined colorimetrically (Method

—351.2). Total phosphorus (TP) was digested with

11 N H2SO4 and potassium persulfate (Method—

365.1). Water samples were filtered through a 0.45 lmmembrane filter and filtrate was analyzed for dissolved

reactive phosphorus (DRP) (Method—365.1), ammo-

nium-N (NH4–N) (Method—351.2), and dissolved

organic carbon (DOC) (automated Shimadzu (Shima-

dzu Corp., Kyoto, Japan) TOC 5050 analyzer

(Method—415.1).

Sediment properties

Sediment samples were transported on ice and stored

in the dark at 4�C. Before each analysis, sampleswere homogenized and sub-samples taken. Sediment

bulk density (g dry wt cm-3 wet) was determined at

70�C for 72 h, and pH was determined on wetsediments (1:2 sediment-to-water ratio). Sediment

samples were ground in a ball mill and passed

through a # 40 mesh sieve. Organic matter content

(LOI-loss on ignition) was determined by weight loss

at 550�C. Total P was analyzed by ignition method,followed by acid digestion (6 M HCl) and measured

colorimetrically (Anderson 1976; Method—365.1,

EPA 1993). Total carbon (TC) and total nitrogen

(TN) were determined using a Flash EA-1121 NC

soil analyzer (Thermo Electron Corporation, Wal-

tham, MA, USA).

Extractable nitrogen (N) and phosphorus (P)

Sediment samples were extracted with 0.5 M K2SO4for extractable N and with 0.5 M NaHCO3 (pH = 8.5)

for extractable P, using a 1:50 dry sediment-

to-solution ratio (Mulvaney 1996; Ivanoff et al.

1998). Extracts from N samples were centrifuged at

10,0009g for 10 min and filtered through Whatman #

42 filter paper. For N analysis, 5 ml of the extracts were

subjected to Kjeldahl nitrogen digestion and analyzed

for total Kjeldahl-N colorimetrically (Method—351.2,

EPA 1993). Digested samples represent total labile

nitrogen (TLN). Undigested N extracts were analyzed

for ammonium (NH4–N) (Method— 351.2, EPA

1993), and represent extractable ammonium (Ext–

NH4–N). The difference between TLN and Ext-NH4-N

represents extractable labile organic nitrogen (LON).

Extracts from P samples were centrifuged at 10,0009g

for 10 min and filtered through a 0.45 lm membranefilter, and analyzed for dissolved reactive P or digested

for TP (with sulfuric acid and potassium persulfate).

Solutions were analyzed by colorimetry, determined by

reaction with molybdate (Method 365.1, EPA 1993).

Undigested P extracts represent labile inorganic

P (LIP), and digested samples represent total labile

P (TLP). The difference between TLP and LIP

represents labile organic phosphorus (LOP).

Extractable and microbial biomass carbon (C)

Microbial biomass carbon (MBC) was measured with

the chloroform fumigation-extraction method (Vance

et al. 1987). Briefly, sediment samples were split in

two: one sample was treated with alcohol-free

chloroform (0.5 ml) to lyse microbial cells, placed

in a vacuum desiccator, and incubated for 24 h. The

duplicate sample was left untreated. Both sets were

extracted with 0.5 M K2SO4 centrifuged at 10,0009g

for 10 min and filtered through Whatman # 42 filter

paper. Carbon extracts were acidified (pH \ 2) andanalyzed in an automated Shimadzu TOC 5050

analyzer (Method—415.1, EPA 1993). Microbial

biomass C was calculated by the difference between

treated and non-treated samples. Extracts from the

untreated samples represent labile organic carbon

(LOC).

Electron donors

Basic catabolic response was characterized by mea-

suring CO2 and CH4 production rates from sediment

samples by addition of different electron donors.

Eight different simple organic compounds (electron

donors) were added separately to each sediment

Biogeochemistry

123

sample. These compounds represent model C sources

commonly present in lake sediments. The C com-

pounds used in the study ranged from amino acids

(alanine and arginine), carboxylic acids (acetate,

formate, butyrate, and propionate), polysaccharide

(glucose), and complex polymeric C (lake suspended

solids, lake-SS). Wet sediment (based on 0.5 g of dry

weight) was added to incubation bottles, sealed with

rubber stoppers and aluminum crimp seals, and

purged with N2 gas to remove oxygen from the

headspace. Alanine, arginine, acetate, formate, buty-

rate, propionate, and glucose were added from

anaerobic sterile stock solutions to sediments on a

C-equivalent basis, reaching a final concentration of

42 mM C (504 lg of C g-1 on a dry weight basis)(Degens 1998). All stock solutions were adjusted to

pH 7.0 (±0.2) using either HCl or NaOH at the time of

preparation to avoid any substrate-pH effects on

microbial communities. The electron donor substrates

were added above background thresholds for each

lake.

Lake-SS was obtained by centrifugation (10,0009g

for 30 min) of water samples collected at approxi-

mately 50 cm depth in the water column of each lake.

At that depth, lake suspended material would have

labile carbon. However, in case of Lake Annie, a

deeper lake, the suspended material at 50 cm depth

was expected to be different from the suspended

material that actually reaches the sediment. In deep

lakes, due to organic matter decomposition and

C-utilization through the water column, the suspended

material that reaches the lake sediments is relatively

more recalcitrant than that found at shallow depths of

the lakes. The intent of this experiment was to verify

the effect of addition of labile carbon present in the

lake suspended material on CO2 and CH4 production

rates.

Lake-SS was characterized for LOI, TC, and TN, as

described previously, and was added on the same

C-equivalent basis (504 lg of C g-1 on a dry weightbasis) as other electron donors. A sample from each

lake-SS was incubated to account for CO2 and CH4production of the material. Values obtained were

subtracted from CO2 and CH4 production rates of the

lake-SS treatment for each lake. Sediments from each

site were also incubated with no substrate addition

(control) to obtain values of basal anaerobic CO2 and

CH4 production rates. Samples were incubated in the

dark under anaerobic conditions at 30�C. Gas samples

were taken at 1, 2, 4, 7, 10, and 14 days and

quantified for CO2 and CH4. Gas samples from Lake

Annie and Lake Okeechobee sediments were also

taken at 20 days of incubation due to low CH4production detected in some treatments at 14 days of

the experiment. Headspace CO2 was measured by gas

chromatography using a Shimadzu GC-8A thermal

conductivity detector at 25�C, and Poropak N columnat 20�C (Supelco Inc., Bellefonte, PA) with He as acarrier gas. Headspace CH4 was analyzed on a

Shimadzu gas chromatograph-8A fitted with flame

ionization detector (110�C), N2 as the carrier gas and a0.3 cm by 2 m Carboxyn 1000 column (Supelco Inc.,

Bellefonte, PA) at 160�C. Prior to measurements ofboth CO2 and CH4, headspace pressure was deter-

mined with a digital pressure indicator (DPI 705,

Druck, New Fairfield, CT). Concentrations of CO2and CH4 produced in samples were determined by

comparison with standard gas concentrations and

production rates were calculated by linear regression

(r2 [ 0.95). Final production rates were determinedafter removing the lag phase (the time between

substrate addition and quantifiable gas production)

in each site.

Statistical analysis

All statistical analyses were conducted with stan-

dardized values of anaerobic CO2 and CH4 produc-

tion rates by microbial biomass carbon of each

sediment sample (CO2 or CH4 production divided by

MBC). One-way analysis of variance (ANOVA) with

pairwise multiple comparisons Tukey’s HSD test was

used to assess the effect of different electron donor

additions on CO2 and CH4 production. One-way

ANOVA was performed separately on each site.

Principal Component Analysis (PCA) was performed

to determine major patterns of CO2 and CH4production rates with the addition different electron

donors. All statistical analyses were conducted with

Statistica 7.1 (StatSoft 2006, Tulsa, OK) software.

Results

Sediment properties

Sediment pH ranged from 5.9 to 7.8 across all locations

(Table 2). Lake Annie sediment pH (5.9) was lower

Biogeochemistry

123

than other lakes. Sediment pH values in both Lake

Okeechobee (M17: 7.7, M9: 7.8 and KR: 7.6) and

Lake Apopka (7.5) were circum-neutral to alkaline

(Table 2). Surface sediment bulk densities were lowest

in Lake Apopka (0.019 g cm-3), followed by Lake

Annie (0.052 g cm-3), and Lake Okeechobee sites

M9 (0.137 g cm-3), M17 (0.143 g cm-3), and KR

(1.183 g cm-3), respectively (Table 2). Sediment

organic matter content was highest at Lake Okeecho-

bee site M17 (86.6%), reflecting high peat content.

Next in order were Lake Apopka (64.9%), Lake Annie

(55.6%), followed by site M9 (37.5%), and sandy KR

(4.6%) in Lake Okeechobee (Table 2). Total C was

highest in peat zone sediments of Lake Okeechobee

(482 g kg-1), followed by sediments from Lake Apo-

pka (288 g kg-1) and Lake Annie (272 g kg-1). Total

N in sediments of Lake Apopka (27.3 g kg-1) and peat

zone (27.7 g kg-1) sediments of Lake Okeechobee

exhibited similar values (Table 2). Lake Annie

(1,427 mg kg-1) sediments exhibited higher TP con-

centrations than Lake Okeechobee (M9: 1,018 g kg-1,

M17: 207 mg kg-1 and KR: 366 g kg-1) and Lake

Apopka (1,185 mg kg-1) sediments (Table 2).

Labile organic C and MBC were highest in Lake

Apopka sediments (4,029 and 42,618 mg kg-1,

respectively) (Table 2). Lake Apopka sediments also

had the highest concentrations of LON (859 mg kg-1)

and Ext–NH4–N (386 mg kg-1) (Table 2). Lake

Annie sediments, however, had the highest concen-

trations of LIP (124 mg kg-1) and LOP (71 mg kg-1)

(Table 2). Sediment LOC:TLN ratio was similar in all

lake sediments (Table 2). Lake Annie and sites M9

and KR in Lake Okeechobee exhibited low LOC:TLP

and TLN:TLP ratios.

Electron donors

Dry suspended material content of the three lakes

(lake-SS) was characterized as: 34% C and 2.9% N

from Lake Annie; 16% C and 1.5% N from Lake

Okeechobee; and 34% C and 3.7% N from Lake

Apopka. Addition of electron donors to sediment

microcosms stimulated heterotrophic microbial activ-

ity in most samples (Tables 3, 4). All sediments

showed a rapid response to addition of electron

donors. Basal CO2 and CH4 production rates were

highest in Lake Apopka sediments (217 mg CO2–C

kg-1 day-1 and 80 mg CH4–C kg-1 day-1) (Tables 3,

4). Sediments from Lake Okeechobee had the longest

lag phase in CH4 production (Data not presented).

Addition of different electron donors produced a

different response in each lake sediment (Table 3).

Results from one-way ANOVAs were significantly

different (P \ 0.05, Table 5). Lake Annie sedimentshad the highest CO2 production rates with both amino

acids (alanine: 217 mg CO2–C kg-1 day-1 and argi-

nine: 212 mg CO2-C kg-1 day-1) and formate

(209 mg CO2–C kg-1 day-1) addition. Lake Oke-

echobee sediments had the highest CO2 production

rates with both amino acids (alanine—M9: 62 CO2-C

kg-1 day-1, M17: 75 CO2-C kg-1 day-1 and KR: 18

CO2–C kg-1 day-1; arginine—M9: 50 CO2–C kg

-1

day-1, M17: 86 CO2–C kg-1 day-1 and KR: 10 CO2–

C kg-1 day-1) and glucose (M9: 66 CO2–C kg-1

day-1, M17: 104 CO2–C kg-1 day-1 and KR: 13

CO2–C kg-1 day-1) addition. Lake Apopka sediments

had the highest CO2 production rates with alanine

(874 CO2–C kg-1 day-1) and formate (773 CO2–C

kg-1 day-1) (Table 3).

Higher CH4 production rates in Lake Annie

sediments were detected with the addition of both

amino acids (alanine: 120 mg CH4–C kg-1 day-1 and

arginine: 159 mg CH4–C kg-1 day-1) and acetate

(155 mg CH4–C kg-1 day-1) (Table 4). In Lake

Okeechobee mud (site M9) sediments, higher CH4production rates were detected in alanine (45 mg

CH4–C kg-1 day-1), butyrate (41 mg CH4–C kg

-1

day-1), and glucose (52 mg CH4–C kg-1 day-1)

treatments. In Lake Okeechobee sand (site KR)

sediments, higher CH4 production rates were detected

in alanine (12 mg CH4–C kg-1 day-1) and glucose

(13 mg CH4–C kg-1 day-1) treatments. In the peat

zone sediments (site M17) of Lake Okeechobee,

addition of the two amino acids (alanine: 10 mg CH4–

C kg-1 day-1 and arginine: 11 mg CH4–C kg-1

day-1), acetate (11 mg CH4–C kg-1 day-1), and

butyrate (8 mg CH4–C kg-1 day-1) produced higher

CH4 than other substrates. In Lake Apopka sediments,

highest CH4 production rates were detected with the

addition of alanine (563 mg CH4–C kg-1 day-1),

glucose (374 mg CH4–C kg-1 day-1), and lake-SS

(355 mg CH4–C kg-1 day-1) (Table 4). The magni-

tude of CO2 and CH4 production following addition of

different electron donors was related to microbial

biomass at each site. There was a strong significant

positive correlation (Pearson’s correlation) between

MBC and CO2 (r = 0.91), and MBC and CH4(r = 0.86) production rates (Fig. 2a, b).

Biogeochemistry

123

Ta

ble

2S

edim

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bio

geo

chem

ical

pro

per

ties

of

Lak

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Biogeochemistry

123

Principal Component Analyses (PCA) of the data

on the effect of electron donor additions on CO2 and

CH4 production rates indicated that 41% of the data

variability was explained by Axis 1 while Axis 2

explained 20% (Fig. 3a). Anaerobic respiration with

the additions of acetate, butyrate, formate, and lake-

Table 3 Sediment anaerobic respiration (mg CO2–C kg-1 day-1) with the addition of different carbon sources

Treatment Anaerobic respiration (mg CO2–C kg-1 day-1)

Lake

Annie Okeechobee Apopka

Central M9 M17 KR West

Basal 100 ± 9a 26 ± 1a 21 ± 4ae 3 ± 2ac 217 ± 23a

Amino acids

Alanine 217 ± 9b 62 ± 7bcd 75 ± 10bcd 18 ± 4bc 874 ± 109b

Arginine 212 ± 10b 50 ± 6bc 86 ± 8bcf 10 ± 4abc 623 ± 350ab

Carboxylic acids

Acetate 135 ± 16a 33 ± 1a 50 ± 10bde 5 ± 5ac 500 ± 259ab

Butyrate 87 ± 3ac 30 ± 2a 33 ± 6ade 4 ± 3ac 307 ± 183ab

Formate 209 ± 22b 28 ± 2a 51 ± 4bde 3 ± 3ac 773 ± 324b

Propionate 55 ± 13c 32 ± 1a 42 ± 11ade 4 ± 3ac 629 ± 152ab

Polysaccharide

Glucose 96 ± 12a 66 ± 5bd 104 ± 25cef 13 ± 3bc 559 ± 129ab

Natural carbon

Lake-SS 101 ± 12a 29 ± 1a 33 ± 3ade 4 ± 2ac 418 ± 160ab

Tukey’s test was conducted within sites and different letters indicate significant statistical differences at P \ 0.05 (mean ± SD)

Table 4 Sediment methane production rates (mg CH4–C kg-1 day-1) with the addition of different carbon sources

Treatment Methanogenesis (mg CH4–C kg-1 day-1)

Lake

Annie Okeechobee Apopka

Central M9 M17 KR West

Basal 37 ± 8a 0.09 ± 0.0a 0.16 ± 0.02a 0.04 ± 0.01a 80 ± 7a

Amino acids

Alanine 120 ± 15b 45 ± 4be 10 ± 4b 12 ± 5b 563 ± 20b

Arginine 159 ± 7c 19 ± 3cdf 11 ± 3b 2 ± 2a 220 ± 24ace

Carboxylic acids

Acetate 155 ± 15c 26 ± 1cd 11 ± 1b 5 ± 9a 102 ± 23acd

Butyrate 85 ± 4d 41 ± 3b 8 ± 1b 5 ± 2a 192 ± 16acd

Formate 36 ± 15a 5 ± 2a 0.3 ± 0.08a 3 ± 4a 44 ± 8acd

Propionate 3 ± 1e 13 ± 1ce 0.8 ± 0.4a 0.4 ± 0.5a 48 ± 10acd

Polysaccharide

Glucose 44 ± 8a 52 ± 5bf 0.7 ± 0.4a 13 ± 5b 374 ± 22ce

Natural carbon

Lake-SS 47 ± 8a 8 ± 2ae 0.0 ± 0.0a 2 ± 1a 355 ± 14ce

Tukey’s test was conducted within sites and different letters indicate significant statistical differences at P \ 0.05 (mean ± SD)

Biogeochemistry

123

SS were the variables selected by Axis 1. Basal

anaerobic CO2 production was selected by Axis 2.

The position of sites in relation to variable loadings in

PCA-1 showed that sediments from each lake and site

are separated into different groups (Fig. 3b). Lake

Annie sediments were plotted in the position of basal

CO2 production (Fig. 3b). Lake Apopka sediments

with lake-SS cluster (lake-SS, butyrate, acetate,

formate, and propionate) opposite from Lake Annie.

Lake Okeechobee site M17 was plotted close to Lake

Apopka sediments, while the KR site was in the

position with glucose and alanine additions. Lake

Okeechobee mud zone (site M9) was not placed with

any specific carbon addition (Fig. 3b).

The PCA-2 had 34% of the data variability

explained by Axis 1 while Axis 2 explained 27%

(Fig. 4a). Methane production rates with additions of

alanine, butyrate, and glucose were the variables

selected by Axis 1. Methane production rates from

arginine, and basal production rate were selected by

Axis 2. The position of the sites in relation to the

variable loadings in PCA-2 showed a separation of

sediments from each lake and site (Fig. 4b). Lake

Annie sediment was placed with the basal production,

arginine, and acetate cluster. Lake Okeechobee M9

site was plotted in the position of propionate and

formate and close to the KR site that was positioned

with glucose, alanine, and butyrate. Site M17 was

plotted in opposite position of all C-sources. Lake

Apopka sediments were placed with lake-SS (Fig. 4b).

Discussion

Addition of organic electron donors to sediments

stimulated heterotrophic activity. Similar results have

also been shown by other studies. Findlay et al. (2003)

showed that the addition of different carbon sources,

i.e., glucose, bovine serum albumin and natural leaf

leachate to hyporheic biofilms enhanced microbial

activities. Wang et al. (2007) showed that addition of

electron donors (glucose, sucrose, potato starch, and

sodium acetate) stimulated denitrification in Lake

Taihu (China) sediments. In the study of benthic

microbial response to the deposition of natural seston

in Lake Erken (Sweden), Törnblon and Rydin (1998)

showed that seston addition caused an immediate

increase in bacterial production, activity, and total

sediment metabolism.

The extent of response to electron donor addition

was related to microbial biomass (Tables 2, 3, 4;

Fig. 2a, b). Generally, sediments responded rapidly to

addition of most electron donors by increasing CO2production rates. Sediments from site KR in Lake

Okeechobee with the lowest microbial biomass exhib-

ited the longest lag phase before responding to electron

donor addition. Microbial biomass was significantly

related to rate of C source consumption in all lake

sediments, suggesting that microbial activity is limited

by C availability (Fig. 2a, b) (Lu et al. 2000).

Although the magnitude of response to electron

donor additions was related to microbial biomass,

Table 5 One-wayANOVA statistics of the

effect of the different

carbon sources addition to

sediment CO2 and CH4production rates

ANOVA Lake

Annie Okeechobee Apopka

Central M9 M17 KR West

Anaerobic respiration (mg CO2–C kg-1 day-1)

n 27 27 27 27 27

df 8 8 8 8 8

F 63.39 33.83 27.36 9.80 3.68

P \0.00001 \0.00001 \0.00001 0.00003 0.0103Methanogenesis (mg CH4–C kg

-1 day-1)

n 27 27 27 27 27

df 8 8 8 8 8

F 91.14 124.30 43.94 4.32 24.34

P \0.00001 \0.00001 \0.00001 0.00471 \0.00001

Biogeochemistry

123

varied responses in sediments were probably related

to catabolic diversity of microorganisms utilizing

these substrates. Results of PCA 1 showed that Lake

Apopka sediments had the highest respiration per unit

of microbial biomass with most of the electron donor

additions (propionate, lake-SS, butyrate, acetate,

and, formate), indicating that these sediments respired

most of the added C (Fig. 3a, b). This suggests higher

microbial activity and catabolic diversity in these

sediments as compared to sediments from the other

lakes. Increased biogeochemical diversity can occur

in environments with high organic matter content due

to the presence of a wide range of organic compounds.

Similar conclusions were made by Castro et al.

0 5 10 15 20 25 30 35 40 450

100

200

300

400

500

600

700

800

Ana

erob

ic R

espi

ratio

n (m

g C

O2-

C k

g-1

d-1 )

Carboxylic AcidsAmino AcidsPolysaccharideLake-SSBasal

r = 0.91

Lake Apopka

Lake Okeechobee

Lake Annie

(a)

0 5 10 15 20 25 30 35 40 45

Microbial Biomass Carbon (g kg-1)

0

50

100

150

200

250

300

350

400

450

Met

hane

Pro

duct

ion

(mg

CH

4-C

kg-

1 d-

1 )

Carboxylic AcidsAmino AcidsPolysaccharideLake-SSBasal

r = 0.86

Lake Apopka

Lake Okeechobee

Lake Annie

(b)

Fig. 2 Relationshipbetween microbial biomass

carbon and activity: a CO2,and b CH4 production rates,with the different groups of

carbon sources added to

sediments from different

lakes

Biogeochemistry

123

(2005), who reported that complete oxidizing species,

capable of using a broader array of electron donors

were dominant in eutrophic and transitional sites of

the Everglades wetlands, while incomplete oxidizers,

that are more efficient in utilizing low concentrations

of fewer substrates, were present in oligotrophic

regions. The authors concluded that eutrophic regions

with high C availability select microbial communities

dominated by generalists that are capable of utilizing a

greater diversity of C substrates (Castro et al. 2005).

Lake Apopka was formed as a result of high primary

productivity in the water column and subsequent

deposition of plankton biomass to benthic sediments

(Gale and Reddy 1994). These sediments support high

rates of microbial activity, as indicated by rapid

utilization of a wide range of electron donors.

Fig. 3 Results of thePrincipal Component

Analysis (PCA-1): aloadings of the effect of

different carbon sources

addition on sediment CO2production rates, and b theplot of the scores of the sites

from Lake Annie (circles),Lake Okeechobee: M9

(squares), M17 (diamonds),KR (crosses), and LakeApopka (triangles). CO2production rates were

normalized by microbial

biomass carbon

Biogeochemistry

123

Alternatively, heterotrophic bacteria tend to respire

most of the added C under conditions of P limitation.

In controlled experiments with bacterioplankton in

subarctic Lake Diktar Erik, Sweden, Jasson et al.

(2006) showed that bacterioplankton respired large

portions of added C under P limiting conditions and

that a significant portion of the added C was also used

to support the growth of microbial biomass.

Addition of some electron donors (i.e., butyrate and

propionate) did not stimulate heterotrophic micro-

bial respiration. With others the stimulation was not

significantly different from basal activities (Tables 3, 4),

Fig. 4 Results of theprincipal component

analysis (PCA-2): aloadings of the effect of

different carbon sources

addition on sediment CH4production rates, and b theplot of the scores of the sites

from Lake Annie (circles),Lake Okeechobee: M9

(squares), M17 (diamonds),KR (crosses), and LakeApopka (triangles). TheCH4 production rates were

normalized by microbial

biomass carbon

Biogeochemistry

123

suggesting either absence of organisms that can utilize

these substrates or assimilation of added C into micro-

bial biomass (Bremer and van Kessel 1990; Degens

1998). Törnblon and Rydin (1998) found that after

seston addition to sediment, bacterial biomass doubled

indicating assimilation of C into microbial biomass. For

forested soils, the partitioning between biomass-C

incorporation and respiratory CO2-C was determined

to be substrate- rather than soil-dependent. van Hees

et al. (2005) reported for forested soils that 60–90% of

organic acid, 20–60% of monosaccharide, and 10–30%

of amino acid is evolved as CO2. Studies with different

microorganisms reported that between 30 and 40% of

glucose and up to 80% of formate of the C source

supplied is immediately used for respiration and the

remaining for biomass growth (Stouthamer 1976). King

and Klug (1982) reported that the addition of glucose

into microbial biomass was low (20%) in a eutrophic

lake sediment (Wintergreen Lake). Results from this

study suggest that a large proportion of added amino

acids, glucose and formate were used through respira-

tory pathways rather than added into biomass (King and

Klug 1982).

PCA results placed Lake Annie sediments with

basal CO2 production rates indicating that this site had

the highest anaerobic respiration per microbial bio-

mass (Fig. 3a, b). Lake Apopka was positioned on the

opposite side, indicating the lowest basal anaerobic

respiration per microbial biomass. The metabolic

quotient (qCO2; proportion of basal respiration per

microbial biomass) has been used in soil studies to

indicate ecological efficiency of the soil microbial

community (Anderson and Domsch, 1990; Degens,

1998). This index is based on Odum’s theory of

ecosystem succession (1969), where during ecosystem

succession towards maturity there is a trend of

increasing efficiency in energy utilization concomitant

with an increase in diversity. High qCO2 indicates

inefficient use of energy, while low qCO2 indicates

high efficiency and more carbon utilized for biomass

production (Anderson and Domsch 1990; Anderson

2003; Francaviglia et al. 2004). Moreover, if the

progression of lakes in time from less productive

(oligotrophic) to more productive (eutrophic-hypereu-

trophic) can be viewed as a natural succession, higher

qCO2 should be detected in oligotrophic lakes. The

trend of decreasing qCO2 with increasing trophic state

is clearly presented in Axis 2 of PCA-1 (Fig. 3a, b).

Similar results were also reported by Smith and Prairie

(2004) in a study of bacterioplankton in lakes of

different trophic states, where they concluded that

oligotrophy places high respiratory demands on bac-

terioplankton, with greater carbon flow to CO2 rather

than to biomass.

Basal CH4 production rates were highest in

sediments of hypereutrophic Lake Apopka. These

results are in accordance with those observed in

other studies that have shown higher CH4 production

rates in sediments from eutrophic lakes when

compared with those from oligotrophic lakes (Falz

et al. 1999; Nüsslein and Conrad 2000; Huttunen

et al. 2003). Low rates of basal CH4 production

found in eutrophic Lake Okeechobee sediments may

be explained by electron donor limitation, or by the

presence of iron oxides which potentially can inhibit

methanogenesis (Roden and Wetzel 2003). Presence

of Fe has been previously documented in Lake

Okeechobee sediments by Fisher et al. (2001). In a

previous study (Torres 2007), basal CH4 production

was not detected, but was stimulated after the

addition of acetate and/or H2 in sediments of Lake

Okeechobee. Although a lag phase for CH4 produc-

tion was observed in all sediments, CH4 production

was much delayed in sediments from sites M17 and

KR in Lake Okeechobee (data not shown). Metha-

nogens have the ability to use only a limited number

of substrates, including H2, CO2, formate, acetate,

methanol, and methylated amines (Oreland, 1988).

The most important substrates for methanogens are

H2/CO2 and acetate, and they often depend on other

anaerobic bacteria for these substrates (Conrad

1999).

Other anaerobic bacteria (i.e., Fe and SO4-2 reduc-

ers) can outcompete methanogens for H2/CO2 and

acetate due to higher substrate affinities and higher

energy and growth yields (Lovley and Klug 1983;

Lovley and Phillips 1986; Bond and Lovley 2002);

however, both processes can coexist (Holmer and

Kristensen 1994; Roy et al. 1997; Roden and Wetzel

2003; Wand et al. 2006). Coexistence occurs because

of spatial variation in the abundance of terminal

electron acceptors or because the supply of electron

donors is non-limiting (Roy et al. 1997; Megonigal

et al. 2004). The lag phase observed for CH4production in all sediments can be explained by two

mechanisms: (1) methanogenic activity stimulated in

the presence of substrates produced by fermentative

activity, and (2) methanogens becoming active after

Biogeochemistry

123

other electron acceptors (Fe(III), SO4-2) were con-

sumed and depleted in sediment microcosms.

Most methanogenic species use H2/CO2 and a fewer

number of species can use acetate (Garcia et al. 2000).

In lake sediments the dominance of acetoclastic

(acetate utilizers) versus hydrogenotrophic (H2 utiliz-

ers) methanogenesis has been reported to be related to

sediment properties (i.e., pH and temperature). In

acidic Lake Grosse Fuchskuhle (Germany), with high

humic content, acetate utilizers (Methanosarcinaceae)

were the only detected methanogens (Casper et al.

2003). Phelps and Zeikus (1984) reported that aceto-

clastic methanogenesis was the major pathway for

CH4 production in a mildly acidic (pH 6.2) lake

(Knaack Lake, Wisconsin). The increase in pH to

neutral values enhanced total CH4 production from H2/

CO2, but did not affect the CH4 produced from acetate

(Phelps and Zeikus 1984). In mesotrophic Lake Rotsee

(Switerland) sediments, it was reported that in these

cold sediments acetate is the main CH4 precursor (Falz

et al. 1999). Other studies have shown that CH4production at low temperature (4�C) in sediments wasmainly from acetate, however, an increase in temper-

ature (20–25�C) lead to an increase in contribution ofCH4 production from H2/CO2 (Schulz and Conrad

1996; Schulz, et al. 1997; Nüsslein and Conrad 2000;

Glissmann et al. 2004).

Lake Annie sediments are acidic and probably

maintain fairly constant low temperatures. Thermal

stratification of the water column was detected during

sampling in this lake with a temperature of 17.3�Cbelow 14 m water column depth (Torres 2007).

Sediment temperature is probably much lower in this

deep (20 m) lake. Sediment acidic pH and low

temperatures as well as the high CH4 production rate

with addition of acetate and the placement of Lake

Annie with acetate cluster in the PCA-2 suggest that

acetoclastic methanogenesis may be an important

pathway for CH4 production in these sediments

(Table 5; Fig. 4a, b). Lake Okeechobee and Lake

Apopka had high temperatures at the sediment–water

surface (26.3–30.7�C), and both lakes had circum-neutral to alkaline sediment pH (Table 2), creating

good conditions for hydrogenotrophic methanogene-

sis. In Lake Okeechobee sediments it has been

determined that hydrogenotrophic methanogenesis is

the main pathway of CH4 production (Torres 2007).

Our results suggest that phytoplankton deposition is an

important source of C to methanogenic activity in

Lake Apopka sediments as evidenced by high rates of

CH4 production with addition of lake-SS (Table 3;

Fig. 4a, b).

In conclusion, this study has shown that sedi-

ments with different biogeochemical properties have

microbial communities that exhibit distinct catabolic

responses to a range of C-sources. The hypereutrophic

lake with higher C availability in its sediments had the

highest catabolic diversity, where the microbial com-

munities were able to efficiently use a broader range

of substrates. The oligo-mesotrophic lake sediment

microorganisms had lowest efficiency in use of energy.

An increasing efficiency in use of C sources by the

sediment microbial community with increasing trophic

conditions was detected. This study establishes a close

linkage between physical and chemical properties of

lake sediments and associated microbial activities.

Acknowledgments This work was funded in part by USDA/NRI grant. We thank the following for their assistance in this

work: Matt Fisher, Jason Smith, Andrea Albertin and Kathleen

McKee for field and sampling assistance, and Yu Wang and

Jeremy Bright for laboratory assistance.

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Heterotrophic microbial activity in lake sediments: effects of organic electron donorsAbstractIntroductionMethodsStudy sitesField samplingSediment propertiesExtractable nitrogen (N) and phosphorus (P)Extractable and microbial biomass carbon (C)Electron donorsStatistical analysis

ResultsSediment propertiesElectron donors

DiscussionAcknowledgmentsReferences

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